P1345: EFFECT OF DECITABINE ON MACROPHAGE PHENOTYPE AND FUNCTION IN VITRO IN THE CONTEXT OF AML
Authors: Jacobien Hilberink, Isabel Weinhäuser, Diego Pereira-Martins, Jan Jacob Schuringa, Gerwin Huls
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Acute myeloid leukemia (AML) is a complex and aggressive disease characterized by a high genetic heterogeneity and clonal diversity across patients. Recent studies have underlined the important contribution of the tumor microenvironment in facilitating a leukemia permissive environment. Targeting this tumor supportive microenvironment can possibly aid in antileukemic treatment and make leukemic cells more susceptible to cytotoxic therapies. Macrophages are important immune cells present in the microenvironment of AML. Due to their high plasticity they encompass both tumor suppressive as well as tumor supportive functions, and are therefore interesting targets. Epigenetic regulation has been suggested to play in role in macrophage polarization. Currently, decitabine (DAC), a hypomethylating agent, is an important component of the armamentarium to treat patients with AML not eligible for intensive chemotherapy. Yet, little is known about the effect of DAC on the leukemic microenvironment. Aims: We decided to evaluate the effect of decitabine treatment on macrophage polarization and functionality. Methods: Monocytes isolated from the peripheral blood of healthy donors were polarized to M1 or M2 macrophage subtypes or kept as M0-unpolarized cells, and subsequently treated with different dosages of DAC (0.1 mM, 0.25 mM, 0.5 mM) for 48 hours to asses phenotypical and functional changes. The successful polarization of macrophages into their respective subtype was determined by flow cytometry for CD80 (M1) and CD163, CD206 and CD200R (M2) marker expression. Results: No change in viability was observed when macrophages were treated with the highest dose of decitabine (0.5 mM), and the percentage and mean fluorescent intensity of M1 and M2 markers remained stable. No dose dependent effect could be observed. Next, cord blood-derived CD34+ cells were co-cultured on different macrophage subtypes treated with vehicle or DAC. On M1 macrophages the proliferation rate of CD34+ was significantly reduced compared to M0 and M2 macrophages. However, no difference was detected between the proliferative support of vehicle and DAC-treated M0/M2 macrophages and neither the tumor suppressive effect of M1 macrophages was altered by the presence of DAC. Also no difference was observed between the distribution of different hematopoietic subpopulations including HSC, LMPP, CMP, GMP and MEP. The same experiment was repeated with primary AML samples, whereby the pre-treatment of M0 and M2 macrophages with DAC resulted in better proliferative support for some AMLs, while in others no difference was observed, probably due to the heterogeneous nature of the disease. Co-culture of AML cells on M1 macrophages pre-treated with either vehicle and DAC led to a strong reduction in AML cell proliferation. Evaluation of cell differentiation markers on AML cells post-coculture indicated a slight increase of CD11b expression when AML cells were co-cultured on M1 macrophages treated with DAC compared to M1 control, while no difference was observed for the other macrophage subtypes. Finally, cord blood-derived CD34+ were differentiated into macrophages in the presence of DAC. DAC treatment led to a significant increase in CD206 (a M2 marker), suggesting that DAC might drive the generation of more immunosuppressive macrophages from stem cells. Summary/Conclusion: Overall our data provide insight into the role of DAC in the generation and functionality of macrophage subtypes which is of relevance for AML patients treated with decitabine and could indicate possible mechanisms that play a role in refractory disease and relapse. Keywords: Macrophage, Hematopoietic microenvironment, decitabine, Human CD34+ cell